Cyclic peptides (CPs) have commercial value as drugs, antimicrobial compounds and antigens in vaccines, but they can be difficult and expensive to produce. Also, the ability to make cyclic peptides of any size and sequence is commercially desirable both for screening of thousands of CPs for biological activity and for the production of specific valuable cyclic peptides.
According to the present knowledge, the so-called homodetic cyclic peptides or homocylopetides, which have a ring composed of amino acids linked by peptide bonds, can be produced by: extraction from natural sources, especially plants, fungi and microbes (Pomilio 2006; Tan 2006; Craik 2007; Cascales 2010; Morita 2010); chemical synthesis (White 2011; Lambert 2001; Davies 2003); cyclization of linear peptide precursors using isolated enzymes (Bolscher 2011; Katoh 2011; Grunewald 2006) including Staphylococcus aureus sortase A (Wu 2011), the Prochloron didemni patG gene product (McIntosh 2010) and trypsin (Thongyoo 2008); and, genetic engineering of various organisms including bacteria and plants, using genes encoding split inteins (Young 2011) and other inteins variants (Katoh 2011; Camarero 2011; Austin 2009), proteases and their homologues and/or cyclic peptide precursors (Katoh 2011; Condie 2011; Donia 2008; Tang 2011; Covello 2010; Schmidt 2010; Schmidt 2007) and non-ribosomal peptide synthetases (Kohli 2001).
Particularly relevant is the production of cyclic peptides based on the process which occurs in plants of the Caryophyllaceae family. It has been shown that in this family, precursor peptides are encoded by DNA (Condie 2011). When a DNA fragment encoding precursors is experimentally expressed in genetically transformed roots of Saponaria vaccaria, for example, a corresponding cyclic peptide is produced in the roots. Similarly, when a chemically synthesized precursor peptide is incubated with extracts of Saponaria vaccaria, a corresponding cyclic peptide is produced.
Also relevant is the use of purified enzymes, especially from recombinant microbes, for in vitro peptide cyclization. Generally these involve the use of chemically synthesized linear peptides which are incubated with a purified enzyme, such as sortase A or the patG gene product, capable of catalyzing the formation of a cyclic peptide from part of the linear peptide.
Existing methods have one or more drawbacks. Extraction from natural sources, especially plants, fungi and microbes is limited by the natural variation and abundance of cyclic peptides from these sources. Depending on the size and composition of the desired CP product, chemical synthesis can be complicated and expensive. Peptide cyclization by sortase A is limited to CP products which include a sorting sequence and usually one or two glycine residues. Production of desired CP product using the split intein method varies widely depending on the sequence. Use of inteins variants usually requires the inclusion of a cysteine in the cyclic product. In vivo peptide cyclization by sortase A is limited to CP products which include a sorting sequence and usually one or two glycine residues. Use of non-ribosomal peptide synthetases generally requires a substrate with a C-terminal thioester moiety.
There remains a need for alternative methods of producing cyclic peptides that overcomes one or more of the drawbacks of the prior art.